Lately the usefulness of a microscope having an auto focus function is becoming recognized in the market place. If the auto focus function of active control, for constantly focusing on a sample, is used, blur and movement of an observation image, generated during a time lapse observation over a long period of time, or when a reagent is administered into a container, can be effectively removed.
There are roughly two types of methods for implementing the auto focus function are roughly divided: the passive method and the active method.
In the case of the passive method, for example, a blur of an observation image is detected based on the change of the position of the objective lens at which the contrast of the observation image of the sample is maximum, and the change amount of this position is fed back to the stage and drive unit of the objective lens, whereby focusing on the sample is maintained. According to this method, not only is a change of the position of the objective lens in the optical axis direction detected, but also a change of the relative positions between the objective lens and the sample in a direction perpendicular to the optical axis direction (hereafter called “plane direction”) is detected, therefore misalignment of the relative positions in the plane direction can be corrected (compensated).
Whereas in the case of the active method represented by a slit projection type, misalignment of the relative positions between an objective lens and a sample in the optical axis direction is detected based on the change of the position of an image of the reflected light of infrared irradiated onto a cover glass holding the sample, hence quick focusing is possible (e.g. Patent Document 1).
In the mean time, as current ultra-resolution technology advances, microscope observation at nanometer precision, which exceeds the resolution of an optical microscope, is receiving attention. However in the case of observation in the resolution range, a micro-change of the observation position of the sample in the observation field of view, generated by the mechanical drift of the apparatus and the thermal expansion of cover glass, which have not been major problems, cannot be ignored. Therefore a technology to maintain the relative positions between the objective lens and the sample in three-dimensional directions is demanded.
Patent Document 1: Japanese Patent Application Laid-Open No. 2008-122857
With the above mentioned prior art, however, if misalignment is generated in the relative positional relationship between the objective lens and the sample, it is difficult to quickly and accurately correct the misalignment.
In the case of the passive method, for example, the change of the relative positions between the objective lens and the sample can be compensated in three-dimensional directions, but if the brightness and contrast of the sample are low, image analysis takes time, and delay or an error occurs in focusing. And in the case of the active method, the change of the relative positions between the objective lens and the sample in the plane direction cannot in theory be detected.